The goal of this proposal is to further our understanding of the enzymology associated with the function of the cytochrome P450 (P450) system as they catalyze the oxidative metabolize of a wide variety of different chemical compounds. Knowledge of the P450s is of fundamental importance to our interpretation of those factors influencing the pharmacological action of drugs, reactions for the initiation of chemical carcinogenesis and maintenance of steroid hormone homeostasis. It is proposed to study the mechanism of electron transfer from the flavin-domain of the flavoprotein, NADPH- P450 reductase to the heme-domain of the hemeprotein, P450 using the technique of electrochemistry recently developed int his laboratory. In addition it is proposed to examine the influence of low oxygen tensions on the function of P450s. Lastly, it is planned to compare the enzymatic properties of orthologous forms of P450c17 that are known to have species-specific activities. To achieve the first goal, a continuous flow spectrophotometric method will be carried out in combination with electrochemical studies to permit measurement of the steady state reduction of flavin and heme components of fusion protein engineered to contain truncated carboxy-termini and mutated flavoprotein domains. Particular interest will focus of a cluster of acidic amino acids associated with the FMN-binding region of the flavoprotein. Further, P450 fusion proteins will be engineered to contain the calmodulin-binding site of nitric oxide synthase (NOS) to determine the role of calcium in regulating electron transport in NOS. The second goal will determine the influence of low oxygen concentrations on the function of P450s using an "oxystat" designed to maintain low oxygen concentrations during a reaction. The affinity of different P450s for oxygen will be determined. The third aspect of this proposal is to determine structure-function relationships when comparing recombinant isozymes of P450c17 catalyzing the 17alpha- hydroxylation of progesterone and pregnenolone. Chimeric proteins will be prepared for rescuing lyase activities of deficient P450c17s.